Cultivating streptomyces desdanus var. desdanus to produce desdanine

ABSTRACT

Microbiological process for preparing the antibiotic desdanine which does not require the addition of an omega -alkylthioAlpha -amino acid to the fermentation medium. Desdanine can be used for preventing rot and spoilage of shell eggs caused by Proteus vulgaris.

United States Patent Malcolm E. Bergy Kalamazoo;

Fritz Reusser, Portage, both of Mich. 30,255

Apr. 20, 1970 Dec. 7, 1971 The Upjohn Company Kalamazoo, Mich.

Inventors Appl. No. Filed Patented Assignee CULTIVATING STREPTOMYCES DESDANUS VAR. DESDANUS T0 PRODUCE DESDANINE 3 Claims, No Drawings [5]] lnt.Cl Cl2d9/00 so FleldolSearch 195/80 561 References Cited UNITED STATES PATENTS 3,364,l l5 1/1968 Mason et al.

Primary Examiner-Joseph M. Golian Anorneys- Roman Saliwanchik and John Kekich C ULTIVATING STREPTOMYCES DESDANUS VAR. DESDANIJS TO PRODUCE DESDANINE BRIEF SUMMARY OF THE INVENTION Desdanine is a useful antibiotic produced by a fermentation process using the micro-organism Streptomyces caelesris, NRRL 2418, wherein an w-alkylthio-a-amino acid is incorporated in the fermentation medium. The production of desdanine by such a process, as well as the recovery and characterization of desdanine is described in U.S. Pat. No. 3,364,115. Desdanine, as described therein, has the formula C H NO; it is a basic antibiotic having a molecular weight of 138; and it is optically inactive.

The microbiological process of the subject invention comprises the use of a novel micro-organism which can produce desdanine in a fermentation medium to which no m-alkylthioa-amino acid has been added. Thus, the subject process eliminates an essential step of the prior art process to prepare desdanine.

DETAILED DESCRIPTION Upon cultivating the novel micro-organism of the subject invention in a fermentation medium, without the addition of an w-alkylthio-a-amino acid, there is obtained the antibiotic desdanine.

Preferably, desdanine is produced when the new micro-organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood also that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include corn steep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurifred ingredients are used as media components.

Production of the compound of the invention can be effected at any temperature conducive to satisfactory growth of the micro-organism, for example, between about 18 and 40 C., and preferably between about 20 and 32 C. Ordinarily, optimum production of the compound is obtained in about 2 to 10 days. The medium normally remains basic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and, in part, on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the micro-organism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant ineflicient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active, vegetative inoculum has thus been secured, it is transfered aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compound, as long as it is such that good growth of the micro-organism is obtained.

THE MICRO-ORGANISM The actinomycete used according to this invention for the production of desdanine is Streptomyces desdanus var. desdanus. One of its strain characteristics is the production of desdanine in a fermentation medium in which no w-alkylthioa-amino acid is incorporated. A subculture of the living organism was deposited without restriction and can be obtained from the permanent collection of the Northern Utilization and Research Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, 111., U.S.A. lts accession number in this repository is NRRL 3814.

The micro-organism of this invention was studied and characterized by Alma Dietz of The Upjohn Research Laboratories.

DESCRIPTION OF THE MICRO-ORGANISM Streptomyces desdanus Dietz, sp. n. (S. desdanus var. desdanus) Color characteristics. White to cream to cream pink aerial mycelium. Melanin negative. Yellow reverse on most media. Appearance on Ektachrome is given in table 1. Reference color characteristics on agar media are given in table 2. The culture may be placed in the Yellow (Y) and Gray (GY) color series of Tresner and Backus [App1. Microbiol. 11:335-338 (1963)] Microscopic characteristics. Straight sporophores (RF) in the sense of Pridham et al. [Appl. Microbiol. 6:52-79 (1958)] bearing small spores (50 or more per chain) by lightmicroscope examination. Spores smooth by direct electronmicroscope examination. Spores treated by the carbon-replica technique of Dietz and Mathews [Appl. Microbiol. 10:258-263 (1962)] show surface ridging when examined with the electron-microscope. Cultural and biochemical characteristics. See table 3. Carbon Utilization. Growth of the culture on carbon compounds was determined by using the synthetic medium of Pridham and Gottlieb [.l. Bacterial. 56:107-114 (1948)] and their modified medium as cited in Shirling and Gottlieb [lnternational Journal of Systemic Bacteriology 16:313-340 (1966)]. In the former, culture growth was good on d-xylose, karab ao t -fw w s Daalaswse s p maltose, sucrose, lactose, cellobiose, raffinose, dextrin, inulin, soluble starch, glycerol, salicin, Na acetate, Na citrate, Na succinate; moderate on the control, rhamnose, dulcitol, D- mannitol, D-sorbitol, inositol, Na formate, Na oxalate, Na tar trate; slight on phenol. There was no growth on cresol or sodium salicylate. In the latter there was slight growth on the plain (negative) control and good growth on the glucose (positive) control. There was strongly positive utilization of L-arabinose, sucrose, D-xylose, D-fructose, raffmose; positive utilization of rhamnose; and doubtful utilization of inositol, D-mannitol, and cellulose. Temperature. Good growth with sporulation occurred at 28-37 C. There was no aerial growth at 18 C., trace aerial growth at 24 C., and no growth at 45 or 55 C. Antibiotic producing properties. The culture produces the antibiotic desdanine. Source. Soil DISCUSSION Streptomyces desdanus, a new species of Streptomyces isolated from soil, produces the antibiotic desdanine. The antibiotic was produced previously, only when methionine, 5- methyl cysteine, or S-ethyl cysteine were added to the fermentation medium of the celesticetin-producing culture, Streptomyces caelesris. S. caelestis is distinctly difierent from S. desdanus in its color characteristics, microscopic characteristics, and general cultural characteristics. A brief comparison of the cultures is given in table 4.

The new soil isolate could not be equated with species of actinomycetes in The Upjohn Collection or in the literature descriptions in Gauze [State Publishing House for Medical Literature, Moscow (1957); English edition translated by F. Danga, The American Institute for Biological Sciences, Washington, DC], Hutter [Systematik der Streptomyceten unter besonderer Berucksichtigung der von ihnen gebildeten 3 ,625,830 3 4 Antibiotica. S. Karger, Basel (Schweiz), New York (1967)], The characteristics of Streptomyces desdanus Dietz, sp. n.,

Krassilnikov [Academy of Sciences, U.S.S.R., Moscow NRRL 3814, are given in the following tables: (1949), English edition translated by J. B. Routien, Chas.

Pfizer & Co., Inc. 1957)], or Waksman [The Actinomycetes, TABLE 1 Vol.2(l96l)]. 5

The distinctive cultural characteristics of the Upjohn soil Appemmlce 01 SlTePhJmI/m Madam Ektflchmme isolate, one of which is its ability to produce the antibiotic Agar medium surface R desdamne, give val dity to the specification of this new soil lso- Creamwmte Bright yellow late as a new species of Streptomyces. in conformity with the Czapaks sucr ..d Do. rules of the international Code of Nomenclature of Bacteria yg lgfffl gff No growth [international Journal Of Bacteriol. i61459-490 (1966)] the 0.1 a tyrosine... Trace cream-whit Do. organism is named and designated as a new species, Strepto- Case f gmf 1 myces desdanus sp. n. it is proposed that this organism, main- 1 llietz A., "Ektachrome Trans arencies as Aids in Actinom eete tamed m h Upjohn Coilecmm as UC 5324' be des'gnated l5 Classiilcation,"Annals 01' the New ork Academy of Sclenoes,60:152 l54, the type strain, Streplamyces desdanus var. desdanus. 1264. v. .N ...id w,. d,

TABLE 2 Reference Color Characteristics of Streptomyces desdcnus ISCC-NBS Method of Color Harmony Manual Designating Colors and a Dic- Agar medium 3rd ed., 1948 tionary of Color Names, 1955 2 Bennett's a light ivory eggshell 89gm pale yellow. S I yell w int yellolwishl-lwhite.

m a a e ow R 2ie squash yellow, maize 87gn ri oder ate yelfiigi P {l file light olive 106gm light olive.

lie dusty olive, light In Do. Czapelr's sucrose S 20a light ivory, eggshell 89gin pale yellow.

bfi yellow tint" 92m yellowish-white. R 210 squash yellow, maize 87gm moderate yellow,

--------------------------- 21a mustard, old g0ld. ....{88gm dark yellow,

94g light olive brown. 3? gm 1 i: ii an 11 51 light Olive E Y 0W B 3" P light mustard tan 8 ll ht olive brown- 106g 1 ght olive. Maltose-tryptone 2dc natural, string 93gm yellowish gray. S 3dc naturai fiba shell pink. pliinlitshnwhili'se.

g -0 ve rown. R zge covert gflege llilgzht glrayiigg alive.

m g ye ow rown. P g t l't g Sugar mogeiilatte yglilggvislh brown.

s 0 ve ay re 0 ve. Yeast extract-malt extract (ISP-2) 2g g gr gm g g S 2ca light ivory, eggshell giigm pae yellow.

in mo erata oran e yellow. R 319 light "{72g dark oran e ye ow. P {ll 51c light olive 106gm light 01 vet lie dusty Olive, light moss green. Do. Oatmeal (ISP-(i) S 3ba pearl, shell tint 102 d t 18h n g 1110 era a green ye 0W. R Wage dusty yellow l05gm girayish greenish yellow. P lie dusty olive light moss greenlotlgm ght olive. Inorganic-salts starch (ISP4) S 3ba pearl, shell tint R.. 3gc light tan 76gm light yellowish brown. I. 2ec biscuit, ecru, oatmeal, sand QOgm grayish yellow. Glycerol-asparagine (ISP-5) S 209. light ivory, eggshell Sggmt pale yellowi h b 7 g s tong ye 0w s rown. R 318 cinnamon, yellowrmaple 5 light yellowish brown,

NOTE.-S=Surface; R=Reverse (all readings from glossy surface of chhi/Ps); P=Pigment.

1 Jacobson, E., W. C. Granville, and C. E. Foss. 1948. Color Harmony anual, 3rd ed. Container Corporation of America, Chicago, Ill.

1 Kelly, K. L., and D. B. Judd. 1955. The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names. U.S. Dept. of Comm. Circ. 553.

... lie dusty olive, light moss green. 106gm light olive.

TABLE 3 Cultural and Biochemical Characteristics of Strepiomyces dcsdanus Medium Surface Reverse Other Agar:

Peptone-iron Trace white Yellow Melanin-negative. Calcium malate White Cream-white to pale yellow. {ggggggggg Glucose-asparagine Ne aerial growth Pale yellow Pale yellow pigment. Skim milk Very slight trace white Yellow gz iggfigfimg Tyrosine White-feathery on edge ..d0 gg gg g gfgfi Xanthine White-leathery; do gggggmggggg Nutrient starch White-feathery on edge do .{g";g g ,fgfg,g gg Yeast extract-malt extract Pale tan-rugose with feathered edge... Deep yellow. Yellow pigment. Bennett's Cream Yellow. Yellow. Czapek's sucrose Cream-tan do Do. Maltose-tryptone Cream-pink Cream-y 110w" Pale yellow. Peptone-yeast extract (ISP6) No aerial growth Yellow ug gig' g f ggg g' .Trrp iss il$ l )..-...-w.:.. ;m m ..do Yellow pigment.

Sporophoretype Straight ..IIII::1::IZIIIIIII:

A E 3 =Cnn1iuued Cultural and Biochemical Characteristics of Streptomyces desdanus Medium Surface Reverse Other @e 1a tifi:

. Yellow pigment. Plain "{Gelatin liquefaction A-ki. Nutrient {Yellow pigment. Broth Gelatin liquefaction M-complcte.

. None to pale yellow to yellow pigment. Synthetic Flecked, yellow surface growth Heavy necked bottom growth- Nitrate not reduced to nitrate. Yellow pigment. Nutrient White aerial on surface growth P001 bottom growth 1151 1itrate not reduced to nitrite. 4 us gmen Litmus milk Blue surface ring "{Pe to iiization.

. A"... .i xfiifi I. .7 l A Comparison of Streptomyces desdtmus and Streptumyces caeleatis Agar medium S. desdanus S. caeleatls Peptone-ironz S.. Trace white No aerial growth. R Yellow Brown. Melanin-negative Melan in-positive. Calcium malate:

S White Trace white. R... 1911881111 Whigs to pale yellow. Ehite. t opgmen opigmen. o Malate not solubilized Malate solubilized. Skim milk:

S Very slight trace white No aerial growth.

gefiom.i "t an irownt e owpgmen....... opgmen. O "{Casein solubilized Casein not solubilized. Tyrosine:

S White-feathery on edge Blue-gray.

Yellow Brown. 0 Yellow pigment Light brown pi ent. Tyrosine solubllized Tyrosine solubi ized. T t Grows at 18-37 0. (poor at 45 C. on Czapeks sucrose)- Grows at 1845 C. (poor at 45 0.).

ampem me -"{No growth at 56 C Fair vegetative growth in 48 hours at 55 0.

Straight to open spiral to spiral.

NoTE.S Surface; R Reverse; O Other characteristics.

Desdanine can be isolated from fermentation medium by the procedures disclosed in US. Pat. No. 3,364,1l5. For example, extraction techniques with water-immiscible solvents can be employed. Suitable such solvents are methylene chloride, l-butanol, lower alkyl alkanoates such as ethyl acetate, butyl, acetate, and amyl acetate, and ketones such as isopropyl butyl ketone. Also, desdanine can be recovered from fermentation medium by adsorption techniques, for ex-' ample, adsorption on cationic exchange resins and elution therefrom. Cationic exchange resins which can be used include both carboxylic acid resins (exemplified by Amberlite lRC-SO and Zeocarb 226) and sulfonic acid resins (exemplified by Dowex 50, Amberlite lR-l20, Nalcite HCR, Chempro C-20, Permutit Q and Zeokarb 225 Further, desdanine can be recovered from fennentation medium by adsorption on. activated carbon, or a resin which is a nonionic macroporouscopolymer of styrene cross-linked with divinylbenzene (exemplified by Amberlite XAD 2 resin-Rohm & Haas Co.), and elution therefrom.

ln a preferred recovery process, desdanine-containing fermentation broth is first filtered to remove mycelia and undissolved solids. Desdanine is then removed from the filtrate by passing the same through a column containing a resin which is a nonionic macroporous copolymer of styrene cross-linked with divinylbenzene. [This resin is prepared by suspension polymerization of styrene divinylbenzene copolymers in the presence of a substance which is a good solvent for the copolymer. See JACS 84, 306 (I962). Suitable resins are known by the trade names Amberlite XAD-l and Amberlite XAD-2.]b The resin is eluted with a solvent system consisting of acetone-water l :l The eluate is concentrated to an aqueous solution, adjusted to a pH of about 6.5 with a mineral acid (hydrochloric acid preferred), and freeze-dried to give a crude preparation of desdanine.

Purification of the above-described crude preparation of desdanine can be accomplished by the procedures disclosed in US. Pat. No. 3,364,l 15. In a preferred purification process, a crude preparation of desdanine, as described above, is purified by silica gel column chromatography to relatively pure crystalline desdanine.

EXAMPLE I Part A. Fermentation A soil stock of Streptomyces desdanus var. desdanus, NRRL 3814, is used to inoculate a series of 500-ml. erlenmeyer flasks, each containing ml. of sterile preseed medium consisting of the following ingredients:

Glucose monohydrate 25 g./liter Pharmamedia' 25 gJliter Tap water q.s. Balance 'Pharrnnmedia is an industrial grade of cottonseed flour produced by Trader's Oil Mill Company, Fort Worth, Tex.

The medium is adjusted to pH 7.2 with aqueous NaOH before sterilization.

The flasks are grown for 3 days at 28 C. on a reciprocating shaker.

Preseed inoculum, described above, is used to inoculate a seed tank containing 20 liters of sterile medium consisting of the following ingredients:

Glucose monohydrate l0 gJliter Comneep liquor l0 g./liter Pharmamedia 2 gJliler Wilson's Peptone Liquor No. I59 I0 g.lliter Lard oil by volume Tap water q.s. Bulunce Part B.

Wilson's Peptone Liquor No. I59 is a preparation of hydrolyzed proteins of animal origin.

The medium is adjusted to pH 7.2 with aqueous sodium I Glucose monohydrate l g./liter I Dextrin 25 gJliter Corn gluten meal 20 gJliter i Oatmeal (Ramon Purina) l5 gJliter Tap water Balance The medium is adjusted to pH 7.2 with aqueous NaOH before sterilization.

The fermentation medium is inoculated with 5 percent (volume/volume) of the seed inoculum, described above. The fermentation proceeds for 2-3 days during which time the fermentation medium is agitated at a rate of 320 r.p.m., and aeration provided at the rate of 250 standard liters/minute at p.s.i. back pressure. The temperature in the fermentation tank is maintained at 28 C. When foaming occurs, sterile lard oil is used as antifoam.

A typical desdanine fermentation, as described above, can be illustrated by the following assay profile:

Assay Days (biounltl/ml.)

- overnight at 32 C.

One biounit (BU) is defined as that amount of the active material which gives a 20 mm. diameter zone of inhibition. One biounit equals about 52 ug. of desdanine. Alternatively, the zone size can be referred to a standard curve prepared with standard desdanine.

Recovery Whole fermentation broth (8.7 liters assaying 500 ugJml. against P. vulgaris), from a desdanine fermentation as described in Part A, is adjusted to pH 10.0 with sodium hydroxide and filtered with the aid of about 5 percent diatomaceous earth. The filter cake is washed with water (500 ml.), andthe cake is discarded. The combined filtrate and cake wash (7.2 liters assaying 420 pig/ml. against P. vulgaris'), is passed (flow rate 20 ml./min.) through a 6.4 cm. diameter column containing 720 ml. of Amberlite XAD-2 resin. The effluent is discarded. The resin is-then eluted with 2 liters of acetone-water l: l The eluate is concentrated to an aqueous solution, adjusted to about pH 6.5 with hydrochloric acid, and freeze-dried; yield, 35 grams of a desdanine preparation assaying 15 ug/mg. against P. vulgaris.

Part C. Purification Silica gel (1 Kg. No. 7734, Merck) is mixed with the solvent system methylene chloride-methanol (95:5) and packed into a 6 cm. (l.D.) glass chromatography column to a constant height of cm. with flowing solvent system.

A preparation of desdanine (25 grams assaying 40 ag/mg. against P. vulgaris), prepared as described in Part B, is mixed with methanol (50 ml.) for 30 minutes and then mixed with 125 g. of silica gel No. 7734. The methanol is removed by evaporation and the dried silica gel-desdanine mixture is added to a small head of solvent system remaining on top of the silica gel column bed. The solvent level is drained to the level of the stating material, fresh solvent system is added, and the column is then washed with 8 liters of this solvent. The solvent system is then changed to methylene chloride-methanol (:10) and the column is developed at a flow rate of 50 ml./min. Fractions (500 ml.) are collected and analyzed by UN. at-232 mp. (methanol). Fractions 24 and 25 are combined and concentrated in vacuo to a volume of 50 ml., filtered, the concentrated further to an oil at which time desdanine crystallizes. The desdanine crystals are suspended in acetone (10 ml.), isolated by filtration, washed with acetone, and dried in vacuo .to a constant weight; yield, 183 mg. of essentially pure crystalline desdanine.

We claim:

1. A process for preparing the antibiotic desdanine which comprises cultivating Srreptomyces desdanus var. desdanus in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of desdanine.

2. A process according to claim 1 which comprises cultivating Streptomyces desdanus var. desdanus in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of desdanine and isolating the desdanine so produced.

3. -A process according to claim 2 in which the isolation comprises filtering the fermentation medium to obtain a filtrate containing desdanine, passing said filtrate through a column containing a resin which is like a nonionic macroporous copolymer of styrene cross-linked with devinylbenzene, eluting said resin with a solvent system consisting of acetone-water l:l to obtain an eluate containing desdanine, concentrating said eluate to an aqueous solution, adjusting the pH of said aqueous solution to about 6.5, and freeze-drying said aqueous solution to give a crude preparation of desdanine.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,625,830 Dated December 7, 1971 Inventor) I Malcolm E. Bergy and Fritz Reusser It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column l, 1 ine 12, For "C H NO"-read C H N O. Column 2, l ine 52', for "28 -37 C." read 28-57%. Column i, I ine 2, after 'tablesz" should read Table 1 Appearance of Streptomyces desdanus on Ektachrome' Table-2 Reference Color Characteristics of Streptomyces desdanus Table 3 Cultural and Biochemical Characteristics of Streptomyces desdanus Table l Comparison of Streptomyces desdanus and Streptomyces caelestis Column 5, l ine 6'7, for "2.]b The" read 2;] The Column 6, line 10, Table 5, for "Nitrate not reduced to nitrate" read Nitrate not reduced to nitrite Column 8, l ine 2H, For "stating" read starting l ine 31, For "the" read then l ine 53, for "wh ich is like a" read which is a Signed and sealed this 11th day of July 1972.

(SEAL) 1 Attest:

I-JDL'IARRI IJ' LE'ICIIER, JR. I ROBERT GOTTiiCI-IALK Attesting Jfl leer Commissioner of Patents F ORM PO-IOSO (IO-69) USCOMM-DC 6O376-P59 U,S, GOVERNMENT PRINTING O FFICE l9! 0-366-334 

2. A process according to claim 1 which comprises cultivating Streptomyces desdanus var. desdanus in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of desdanine and isolating the desdanine so produced.
 3. A process according to claim 2 in which the isolation comprises filtering the fermentation medium to obtain a filtrate containing desdanine, passing said filtrate through a column containing a resin which is a non-ionic macroporous copolymer of styrene cross-linked with divinylbenzene, eluting said resin with a solvent system consisting of acetone-water (1:1) to obtain an eluate containing desdanine, concentrating said eluate to an aqueous solution, adjusting the pH of said aqueous solution to about 6.5, and freeze-drying said aqueous solution to give a crude preparation of desdanine. 